Project 1. Construction of the new parts in the SubtiToolKit (STK) syntax

Goal:

Thanks to the kind support of prof. Caro-Astorga, we received access to 96 different DNA parts functionable in B. subtilis. To facilitate the assembly of the full transcriptional units (TUs), we needed to produce our parts fused with appropriate overhangs, allowing the creation of DNA ends compatible with STK upon digestion with respective type IIS restriction enzymes.

Graphic abstract

Assembly of the new parts in the SubtiToolKit (STK) syntax

Results

We ordered the sequences of the Parts fused with respective overhangs, from IDT or Twist. Our first step was the resuspension of the Parts according to the producer’s guidelines. Next, the parts were either cloned directly to the entry vector pSTK-0-GFP through the Golden Gate assembly reaction with BbsI enzyme or first amplified with Q5 polymerase and cloned in the same reaction. White colonies (without the dropout GFP cassette) were first verified using colony PCR. Next, plasmids from the positive colonies were isolated and prepared for sequencing. In the end, we constructed level 0 plasmids containing codon-optimized sequences of: phoA, AMPs, SUMO and thioredoxin.

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Project 2. Benchmark growth experiments

Goal:

The primary goal of these experiments is to systematically evaluate different agar media to identify the most suitable conditions for the growth of Bacillus subtilis and Phytophthora capsici and P. palmivora. This step is essential for our downstream competition assays, where our transformed B. subtilis will be tested for its ability to inhibit Phytophthora growth. We essentially want to test microbial growth as close as possible to the competition assay conditions. Additionally, we performed a calibration experiment to establish a correlation between optical density (OD₆₀₀) measurements and the actual colony forming unit (CFU) counts of B. subtilis. This calibration allows us to translate OD readings into reliable estimates of bacterial concentration for use in later experiments.

Graphic abstract

Benchmark growth assay

Results

We repeated the assay thrice, each time including 4 wells with a particular organism incubated on a particular media. Then, we used the analysis tool of the Reshape Biotech Imaging Device to measure microbial growth over time. Based on the results, we chose PSA as a medium for the competition assays.

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Project 3. Set up of a secretion tag reporter assay in B. subtilis

Goal:

The goal of this assay was to engineer and test the constructs, in which the production of alkaline phosphatase fused with different secretion tags is induced by xylose - analogous to the AMP-containing constructs.

Graphic abstract

Alkaline phosphatase secretion assay

Results

For the pilot assay, we assembled constructs with phoA fused with two different secretion tags: SacB and Apr, or a Cx linker. We used two different RBS sequences in the assembly - a strong RBS used frequently with the popular Phyperspank promoter, or an RBS equipped with the sequence stopping the translation in E. coli to prevent potential toxicity. In the end, we obtained fully correct sequences of the constructs with Apr secretion tag, Cx linker, and SacB with the RBS preventing translation in E. coli.
In the assay, we employed both positive (a purified alkaline phosphatase enzyme from Promega) and negative controls (samples from wild type B. subtilis 168). We tested supernatant and cell lysate samples from all B. subtilis strains. In the end, we saw clear, reproducible signal from the positive control, little background signal from the negative controls, and clear signal from the Apr construct assembled with the RBS blocking translation in E. coli.

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Project 4. Engineering of B. subtilis strains expressing AMPs

Goal:

The aim of the main project of the wet lab team was the construction of plasmids allowing for the production and secretion of antimicrobial peptides by B. subtilis , using parts developed by Caro-Astorga et al. 2025, as well as basic parts incorporated into STK syntax by our team in the previous weeks.

Construction and evaluation of B. subtilis strains expressing AMPs

Results

After many rounds of the optimization of the GGA cloning, we obtained the following constructs:

• Plasmids encoding for the vicilin-like antimicrobial peptide 2-2 with both Cx linker and SacB secretion tag
• Plasmid encoding for MIAMP 2-2 with Cx linker
• Plasmids encoding for cecropin with both Cx linker and SacB secretion tag

B. subtilis strains transformed with the above-mentioned plasmids were tested for their ability to inhibit Phytophthora growth in the dual culture assay established in the previous weeks. While we did not see a significant inhibitory effect, our experimental pipeline can be re-used in the future for the development of reliable assays measuring fungal or oomycete growth inhibition.

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Project 5. Purification of SUMO-AMP fusions

Goal:

The versatility of our cloning approach allows us to seamlessly switch between cloning of the secretion tag-AMP fusions to purification tag-AMP fusions, just by exchanging one DNA part in the Golden Gate assembly. The goal of this project was to assess the efficiency of the cloning and purification of SUMO-tagged AMPs produced by B. subtilis.

Plan for the purification of SUMO-AMP fusion proteins

Results

After a few rounds of the optimization of the GGA cloning, we obtained a plasmid allowing for the expression of the SUMO-AMP03 (cecropin) fusion (described in the entry related to “Construction of E.coli - B. subtilis shuttle vectors for the expression of AMPs”). We transformed B. subtilis with the construct but struggled to obtain the sufficient growth of the engineered strain.

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